Rna containing coenzymes, biotin, or fluorophores, and methods for their preparation and use

ABSTRACT

Materials and methods for incorporating adenosine derivatives into the 5′ end of transcribed RNA are disclosed. Adenosine derivatives include naturally occurring compounds such as Coenzyme A, NAD, and FAD, as well as various non-naturally occurring compounds. The derivatives can be used to impart desirable properties to the RNA such as fluorescence, the ability to bind to receptors or ligands, and improved catalytic activity. The transcribed RNAs can be used in a variety of applications including nucleic acid detection, designed or random generation of catalytic RNAs, antisense applications, and in the study of RNA structure and function

FEDERAL RESEARCH STATEMENT

[0001] The government may own rights in the present invention pursuantto grant number MCB9974487 from the National Science Foundation andgrant number NAG5-10668 from the National Aeronautics and SpaceAdministration.

BACKGROUND OF INVENTION

[0002] 1. Field of the Invention

[0003] The invention relates to RNA molecules having adenosinederivatives incorporated into the 5′ end, and methods and materials fortheir preparation and use.

[0004] 2. Description of the Related Art

[0005] Coenzymes are a group of small organic molecules with distinctchemical functionalities, which are usually unavailable or insufficientin the side chains of the 20 naturally occurring amino acids. A numberof metabolic protein enzymes require coenzymes to act as accessorymolecules to provide necessary reactivities.

[0006] Three common coenzymes, coenzyme A (CoA), nicotinarmide adeninedinucleotide (NAD), and flavin adenine dinucleotide (FAD) carry out avariety of acyl group transfer and electron/hydride transfer reactionsin metabolism. Although CoA, NAD, and FAD function in very differentchemical capacities, they share some surprisingly common structuralfeatures: a ribonucleoside adenosine at one end, a chemically functionalgroup (pantetheine, nicotinamide, or riboflavin) at the other, and apyrophosphate connecting the two groups.

[0007] Because the structures and functions of these coenzymes areconserved in all kingdoms, CoA, NAD, and FAD are believed to haveexisted since the last common ancestor of life on Earth. Furthermore,the fact that all these coenzymes contain an adenosine group, one of thefour essential building monomers for RNA, suggests the plausibility oftheir existence and biological function in a more ancient world.Recently, RNA-catalyzed synthesis of these three coenzymes from theircorresponding precursors, phosphopantetheine, nicotinamidemononucleotide (NMN), and flavine mononucleotide (FMN) (4) wasdemonstrated (Huang, F., et al. Biochemistry39: 15548-15555 (2000)),providing strong experimental evidence of possible coenzyme synthesisand utilization in the RNA world (Gilbert, W. Nature 319: 618 (1986)).

[0008] Currently known ribozymes, either naturally occurring or isolatedin vitro, however, do not utilize these coenzymes to perform chemistriesas protein enzymes do. A recent report has demonstrated RNA-catalyzedacyl CoA synthesis (Jadhav, V. R., and Yarus, M. Biochemistry41: 723-729(2002)), and ribozymes have been isolated capable of catalyzingthioester formation (Coleman, T. M., and Huang, F. Chem. Biol., 9:1227-1236 (2002)). Yet, RNA catalytic activities involving NAD and FADredox chemistry have not been shown to be within the functional capacityof RNA. Since the development of in vitro selection (SELEX) (Ellington,A. D., and Szostak, J. W. Nature 346, 818-822 (1990); Tuerk, C., andGold, L. Science 249: 505-510 (1990); Robertson, D. L., and Joyce, G. F.Nature 344: 467-468 (1990)), numerous artificial ribozymes have beenisolated from random RNA libraries (Jaschke, A. Curr Opin. Struct. Biol11: 321-326 (2001)).

[0009] Convenient and efficient in vitro transcription methods haveplayed an important role in the advancement of RNA research by providingeasily available RNA with defined sequences. However, all the currentmethods require a guanosine derivative as the transcription initiatorunder T3, T7, and SP6 promoters (Milligan, J. F., et al., Nucleic AcidsRes. 15: 8783-8798 (1987); Pokrovskaya, I. D., and Gurevich, V. V., AnalBiochem. 220: 420-423 (1994)). Although other nucleotides such asadenosine and cytidine derivatives have been shown to be able toinitiate transcription with certain sequences (Nam, S. C., and Kang, C.W., J. Biol. Chem. 263: 18123-18127 (1988); Helm, M., et al., RNA 5:618-621 (1999)), the initiation sites are not well defined. The yieldsare low (Nam, S. C., and Kang, C. W., J. Biol Chem. 263:18123-18127(1988)), and they usually result from non-templated incorporation oromission of one or two nucleotides (Helm, M., et al., RNA 5: 618-621(1999)).

[0010] Development of methods for the preparation of RNA moleculeslinked to coenzymes and other adenosine derivatives may provideinteresting catalysts that have improved or novel properties as comparedto current RNA catalysts. Incorporation of fluorophores or biotin at thespecific 5′ end of RNA results in site-specifically labeled RNA that maybe used in RNA structural and functional investigation and nucleic aciddetection.

SUMMARY OF INVENTION

[0011] Chemical and enzymatic methods for the preparation of RNAmolecules covalently linked to coenzymes or other adenosine derivativesare disclosed. An in vitro transcription method has been developed thatprovides for the covalent incorporation of derivatives into the 5′ endof a transcribed RNA molecule.

BRIEF DESCRIPTION OF DRAWINGS

[0012] The following figures form part of the present specification andare included to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these figures in combination with the detailed description ofspecific embodiments presented herein.

[0013]FIGS. 1A-1B shows the structure of adenosine (I), Coenzyme A (II),NAD (III), and FAD (IV).

[0014]FIG. 2 shows the structure of an adenosine 5′-((Ï

-amino-linker) phosphoramidate (V) and an N6-(Ï

-amino-linker) adenosine 5′-monophosphate (VI).

[0015]FIG. 3 shows the production of RNA transcripts incorporatingadenosine derivatives using the T7 Class II RNA Polymerase promoter(Î¦2.5). R-A represents an adenosine derivative; NTPs represents amixture of ATP, UTP, CTP, and GTP. The top sequence is SEQ ID NO:7, andthe bottom sequence is SEQ ID NO:9.

[0016]FIGS. 4A-4C shows the concentration effects of RNA transcriptionin the presence of CoA (FIG. 4A), NAD (FIG. 4B), and FAD (FIG. 4C).

[0017]FIG. 5 shows a method of synthesizing adenosine 5′-(Ï

-amino-linker) phosphoramidate.

[0018]FIG. 6 shows a method of synthesizing N6-(Ï

-amino-linker) adenosine 5′-monophosphate.

BRIEF DESCRIPTION OF SEQUENCES

[0019] The following sequence listings form part of the presentspecification and are included to further demonstrate certain aspects ofthe present invention. The invention may be better understood byreference to one or more of these sequences in combination with thedetailed description of specific embodiments presented herein.

[0020] SEQ ID NO:1 is the CoES7 ribozyme sequence.

[0021] SEQ ID NO:2 is a DNA duplex template oligonucleotide.

[0022] SEQ ID NO:3 is a DNA duplex template oligonucleotide.

[0023] SEQ ID NO:4 is a RNA sequence transcribed from the DNA duplextemplate.

[0024] SEQ ID NO:5 is a DNazyme sequence.

[0025] SEQ ID NO:6 is a T3 promoter sequence.

[0026] SEQ ID NO:7 is a T7 class 11 promoter sequence.

[0027] SEQ ID NO:8 is a SP6 promoter sequence.

[0028] SEQ ID NO:9 is the reverse complement of SEQ ID NO:7.

DETAILED DESCRIPTION

[0029] Currently known ribozymes are RNA molecules selected for theirability to bind a substrate, to catalyze a reaction, or to perform someother selectable activity. In order to use coenzymes (as do many proteinenzymes), the RNA molecules would have to additionally bind thecoenzyme(s). Covalent coupling of coenzymes to RNA sequences not onlyeliminates the requirement of coenzyme binding by ribozymes, but mayalso provide a practical means of selection based on the coenzymereactivity. The development of a general method of incorporating CoA,NAD, and FAD into RNA through in vitro transcription is disclosedherein. This procedure may be used to construct coenzyme-linked RNAlibraries for in vitro selection or to prepare coenzyme-coupled specificRNA sequences for other purposes, such as fluorescent labeling anddetection of RNA/DNA, investigation of RNA structure, and studyingRNA-RNA interaction and RNA-protein interaction.

[0030] RNA transcription can be initiated by adenosine derivatives,including ATP, De-P-CoA, NAD, and FAD using a transcription promotersequence derived from T7 class 11 promoters. Adenosine can be used toprepare adenosine-initiated RNA with free 5′ hydroxyl groups, permittingeasy ³²P-labeling of the 5′ end of RNA by polynucleotide kinase. Becauseonly the adenosine group is required for recognition by T7 RNApolymerase to initiate transcription, a large number of adenosinederivatives can be used to prepare adenosine derivative-linked RNA. Inaddition to the three coenzymes (CoA, NAD, and FAD) and variousadenosine derivatives described in the Examples below, a variety ofbiologically active molecules can be linked to the 5′-end of RNA. Theseinclude coenzymes S-adenosylcysteine, S-adenosylhomocysteine (AdoHcy),and S-adenosylmethionine (SAM), sugar-containing molecule adenosine5′-diphosphoglucose (ADPG), and the signaling molecules diadenosinepolyphosphates Ap(3)A and Ap(4)A. Chemically modified adenosinederivatives such as fluorescent ATP derivatives may also be used toprepare 5′ fluorophore-linked RNA. Similar strategies have been used toprepare 5′ fluorophore-linked RNA by guanosine derivatives. While otherRNA polymerases such as E. coli RNA polymerase can be used to prepareadenosine derivative-initiated RNA, the well defined T3, T7, and SP6promoters, high yields of transcription, and easy preparation of thesephage RNA polymerases make them attractive for in vitro RNA preparation.

[0031] ATP and adenosine derivatives can initiate transcription of anydefined sequences with high efficiency, thereby providing researcherswith choices of different 5′-ends of RNA. High yields of adenosinederivative-initiated random RNA libraries can be achieved, reaching ashigh as 100 mg RNA per 100 mL transcription under standard transcriptionconditions in preliminary experiments. Up to 300 copies of 50 nt RNAmolecules can be synthesized from a single DNA template according topreliminary results.

[0032] In transcription where an adenosine derivative (R-A) other thanATP is present, both normal RNA (pppRNA) and adenosine derivative-linkedRNA (R-ARNA) are produced. Although high concentrations of adenosinederivatives (such as De-P-CoA, NAD, and FAD) lead to high relativeyields of R-RNA over total RNA (pppRNA and R-RNA), total RNA yields maydecrease at high concentrations of adenosine derivatives. To balance thetotal RNA yields and relative yields of adenosine derivative-linked RNA,transcription in the presence of 1 mM each of the four nucleosidetriphosphates and 4 mM of an adenosine derivative can be performed.However, if higher relative yields of adenosine derivative-linked RNAare desired and lower total RNA yields are acceptable, higherconcentrations of adenosine derivatives or lower concentrations of ATPmay be used. At higher than 8 mM of adenosine derivative concentrations,total RNA yields can be significantly lower. At 0.2-0.5 mM ATP and 4 mMDe-P-CoA, transcription produces over 50% CoA-RNA with about 20-50%reduction in total RNA yields. Optimization of transcription conditionssuch as magnesium concentration may improve the total RNA yield andrelative yield of adenosine derivative-linked RNA.

[0033] The fluorescence property of FAD-RNA can be used to investigateRNA structure and function relationship. The defined 5′ end location ofthe fluorophore FAD and easy preparation of FAD-RNA by the in vitrotranscription methods disclosed herein should be advantageous forstructural investigation of RNA by steady-state fluorescencespectroscopy. The lifetime of the fluorophore FAD in FAD-RNA may changeand correlate with its chemical environment, thereby providing a sensorfor RNA folding and conformation through time-resolved fluorescencespectroscopy. Furthermore, another fluorophore (either a donor or anacceptor) can be introduced to other locations in the RNA to allowinvestigation of spatial arrangement of the RNA around its 5′ end byfluorescence resonance energy, transfer (FRET). For small RNA fragments,fluorophores can be added at specific locations by chemical synthesis.However, introduction of a specific chemical group (including afluorophore) at a defined location in relatively large RNA may involvemultiple steps of transcription, chemical synthesis, and ligation. Withan appropriate fluorescence donor or acceptor, FAD-initiatedtranscription can significantly reduce the number of steps needed toprepare RNA suitable for a broad range of FRET studies.

[0034] FAD-RNA may find other applications such as fluorescencedetection of complementary RNA/DNA sequences. 5′ FAD-labeled differentRNA sequences can be conveniently synthesized by the in vitrotranscription methods disclosed herein, to allow detection of largenumber of different RNA/DNA sequences.

[0035] In addition, the reduced form of NAD (i.e., NADH) is also highlyfluorescent. Therefore, NADH-RNA may find wide range of applications asdiscussed above. NADH and FAD have different spectral properties andlifetimes and may respond differently to their RNA environment. FAD-RNAand NADH-RNA may accordingly be combined in RNA structural investigationby fluorescence spectroscopy.

[0036] Aspects of the invention include nucleic acid molecules, methodsfor their preparation, and methods for their use.

[0037] Nucleic Acid Molecules

[0038] One embodiment of the invention includes RNA molecules containingadenosine derivatives covalently attached-to the 5′ end of the RNAmolecule. The RNA molecule can generally have any RNA sequence. Thelength of the RNA sequence can generally be any number of bases. Forexample, the RNA sequence can be at least about 5 bases, at least about10 bases, at least about 20 bases, at least about 30 bases, at leastabout 40 bases, at least about 50 bases, at least about 60 bases, atleast about 70 bases, at least about 80 bases, at least about 90 bases,at least about 100 bases, at least about 200 bases, at least about 300bases, at least about 400 bases, at least about 500 bases, at leastabout 600 bases, at least about 700 bases, at least about 800 bases, atleast about 900 bases, at least about 1000 bases, at least about 2000bases, and so on. There is no practical upper limit to the length of theRNA sequence, as it is dependent primarily upon the length of thetemplate nucleic acid sequence used in its preparation (or upon thesynthetic method used). The RNA molecule can be purified, isolated, orcan be present as a mixture of RNA molecules.

[0039] The adenosine derivative can generally be any adenosinederivative, including coenzymes. Examples of the adenosine derivativesinclude coenzyme A (“CoA”), nicotinamide adenine dinucleotide (“NAD”),flavin adenine dinucleotide (“FAD”), S-(5′-adenosyl)-cysteine,S-(5′-adenosyl)-homocysteine, S-(5′-adenosyl)-methionine, adenosine5′-diphosphoglucose, adenosine 5′-(Î±,{circumflex over (I+EE²-methylene)diphosphate, adenosine 5′-({circumflex over(I)})}²,Î³-imido)triphosphate, adenosine 5′-triphosphateÎ³-(1-(2-nitrophenyl)ethyl) ester, and5′-p-fluorosulfonylbenzoyladenosine. The adenosine derivatives can bemodified at the 5′ position, the N6 position, or both.

[0040] The adenosine derivative can include additional chemicalfunctional groups that can be used for further post-transcriptionalmodification.

[0041] For example, the adenosine derivative may have a thiol group(—SH), an amine group (—NH₂, —NHR, —NR₂), a phosphorothioate group(—O—P(S)O₂), a carboxylic acid group (—CO₂H), a hydroxyl group (—OH), acarbonyl group (C═O), an aldehyde group (—CHO), an amide group (CHN), ora protected functional group, any of which could be used in furtherchemical reactions.

[0042] Additional modifications can include covalently attaching anamino acid, peptide, or protein to the transcribed RNA molecule. Theattaching step is presently preferred to include the formation of anamide bond between a primary amine group in the adenosine derivative anda carboxylate group in the amino acid, peptide, or protein. Theattaching step can be performed on the adenosine derivative prior to itsincorporation into the transcribed RNA molecule, or after itsincorporation. It is presently preferred that the attaching step beperformed on the adenosine derivative prior to its incorporation intothe transcribed RNA molecule.

[0043] Adenosine derivatives can generally be any adenosine derivativesuch as a coenzyme. Examples of coenzymes include coenzyme A (“CoA”;C₂₁H₃₆N₇O₁₆P₃S), nicotinamide adenine dinucleotide (“NAD”;C₂₁H₂₇N₇O₁₄P₂), flavin adenine dinucleotide (“FAD”; C₂₇H₃₁N₉O₁₅P₂Na₂),S-(5′-adenosyl)-cysteine (C₁₃H₁₈N₆O₅S), S-(5′-adenosyl)-homocysteine(C₁₄H₂₀N₆O₅S), S-(5′-adenosyl)-methionine (C₁₅H₂₃N₆O₅SCl, chloride),adenosine 5′-diphosphoglucose (C₁₆H₂₃N₅O₁₅P₂Na₂) adenosine5′-(Î±,Î²-methylene)diphosphate (C₁₁H₁₇N₅O₉P₂), adenosine5′-(Î²,Î³-imido)triphosphate (C₁₀H₁₇N₆O₁₂P₃), adenosine 5′-triphosphateÎ³-(1-(2-nitrophenyl)ethyl) ester (C₁₈H₂₃N₆O₁₅P₃),5′-p-fluorosulfonylbenzoyladenosine (C₆H₄FN₃O₃S), adenosine5-O-thiomonophosphate C₁₀H₁₂N₅O₆P), adenosine 5′-[Î²-thio] diphosphate(C₁₀H₁₂N₅O₉P₂S), and adenosine 5′-[Î³-thio] triphosphate(C₁₀H₁₂N₅O₁₂P₃S).

[0044] The adenosine derivatives can be modified at the 5′ position, theN6 position, or both. Adenosine derivatives can generally be prepared byany acceptable synthetic chemical method or enzymatic method.

[0045] Adenosine 5′-(Ï

-amino-linker) phosphoramidate molecules can be prepared by contacting adiamine, AMP, and EDAC. The diamine can generally be any diamine.Diamines include ethylenediamine (EDA), 1,4-butanediamine (BDA),1,6-hexanediamine (HDA), 2,2′-oxydiethylamine (ODEA),2,2′-(ethylenedioxy)diethylamine (EDODEA), and4,7,10-trioxa-1,13-tridecanediamine (TOTDDA). These diamines providelinkers of (CH₂)₂, (CH₂)₄, (CH₂)₆, C₂H₄OC₂H₄, and C₃H₆OC₂H₄OC₂H₄OC₃H₆,respectively. Additional diamines (and polyamines) such as spermidine(NH₂ (CH₂)₃NH(CH₂)₄NH₂), spermine, (NH₂(CH₂)₃NH(CH₂)₄NH(CH₂) ₃NH₂),polyethylene glycol diamines (H₂N-PEG-NH₂),1,4-bis(3-aminopropoxy)butane (H₂N(CH₂)₃O(CH₂)₄O(CH₂)₃NH₂),bis(3-aminopropyl)amine (H₂N(CH₂)₃NH(CH₂)₃NH₂), tris(2-aminoethyl) amine(N(CH₂CH₂NH₂)₃), 1,8-diaminooctane (H₂N(CH₂)₈NH₂), 1,10-diaminodecane(H₂N(CH₂)₁₀NH₂), and 1,12-diaminododecane (H₂N(CH₂)₁₂NH₂), can also beused.

[0046] These adenosine 5′-(Ï

-amino-linker) phosphoramidate molecules can be generically representedby structure (V) in FIG. 2. In the structure, linker L can genericallybe any alkyl, alkoxy, alkoxy alkyl, alkylamine, or polyalkylamine group.Specific examples include adenosine 5′-aminoethyl phosphoramidate(5′-EDA-AMP), adenosine 5′-(4-aminobutyl) phosphoramidate (5′-BDA-AMP),adenosine 5′-(6-aminohexyl) phosphoramidate (5′-HDA-AMP), adenosine5′-(2-aminoethoxyethyl) phosphoramidate (5′-ODEA-AMP), adenosine5′-(3,6-dioxa-8-aminooctyl) phosphoramidate (5′-EDODEA-AMP), andadenosine 5′-(4,7,10-trioxa-13-aminotridecyl) phosphoramidate(5′-TOTDDA-AMP).

[0047] Adenosine 5′-(Ï

-sulfhydryl-linker) phosphoramidate molecules can be prepared bycontacting an aminothiol, AMP, and EDAC. The aminothiol can generally beany molecule containing a free amino group and a free sulfhydryl group.Aminothiols include cysteamine and carboxyl group-protected cysteine.

[0048] The adenosine 5′-(Ï

-amino-linker) phosphoramidate molecules can be further reacted with anactivated group to modify the linker terminal amino group. For example,succinimide esters can be used to add additional functional moieties.One example of this concept is reaction with 5(6)-carboxyfluoresceinN-hydroxysuccinimide ester (5(6)-FAM-SE) to produce fluoresceinderivatives. Carboxyfluorescein is commonly obtained as a mixture of 5-and 6- isomers, although either can be used individually. A list ofexample products includes 5′-FAM-EDA-AMP, 5′-FAM-BDA-AMP,5′-FAM-HDA-AMP, 5′-FAM-ODEA-AMP, 5′-FAM-EDODEA-AMP, and5′-FAM-TOTDDA-AMP. Similarly, biotin N-hydroxysuccinimide ester(biotin-SE) can be used to produce biotinylated derivatives. A list ofexample products include 5′-biotin-HDA-AMP, 5′-biotin-EDODEA-AMP, and5′-biotin-TOTDDA-AMP.

[0049] The adenosine 5′-(Ï

-sulfhydryl-linker) phosphoramidate molecules can be further reactedwith an activated group to modify the linker terminal thiol group. Forexample, haloacetamide, halide, and maleimide derivatives can be used toadd additional functional moieties. One example of this concept isreaction with fluorescein maleimide, or fluorescein bromoacetamide toproduce fluorescein derivatives of adenosine. Biotin derivatives ofmaleimide or haloacetamide can be used to produce biotinylatedderivatives of adenosine.

[0050] Adenosine thiophosphate molecules (adenosine5′-O-thiomonophosphate, adenosine 5′-[Î²-thio]diphosphate, adenosine5′-[Î³-thio]triphosphate) can also be reacted with an activated group tomodify the thiophosphate group. For example, haloacetamide, halide, andmaleimide derivatives can be used to add additional functional moieties.One example of this concept is reaction with fluorescein maleimide, orfluorescein bromoacetamide to produce fluorescein derivatives ofadenosine. Biotin derivatives of maleimide or haloacetamide can be usedto produce biotinylated derivatives of adenosine.

[0051] N6-(Ï

-amino-linker) adenosine 5′-monophosphate can be prepared by contacting6-chloropurine riboside first with POCl3 then water to produce6-chloropurine riboside 5′-monophosphate. This intermediate can bereacted with a diamine to produce an N6-(Ï

-amino-linker) adenosine 5′-monophosphate. Diamines includeethylenediamine (EDA), 1,4-butanediamine (BDA), 1,6-hexanediamine (HDA),2,2′-oxydiethylamine (ODEA), 2,2′-(ethylenedioxy)diethylamine (EDODEA),and 4,7,10-trioxa-1,13-tridecanediamine (TOTDDA). These diamines providelinkers of (CH₂)₂, (CH₂)₄, (CH₂)₆, C₂H₄OC₂H₄, and C₃H₆OC₂H₄OC₂H₄OC₃H₆,respectively. Additional diamines (and polyamines) such as spermidine(NH₂(CH₂)₃NH(CH₂)₄NH₂), spermine, (NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NH₂),polyethylene glycol diamines (H₂N-PEG—NH₂),1,4-bis(3-aminopropoxy)butane (H₂N(CH₂)₃O(CH₂)₄O(CH₂)₃NH₂),bis(3-aminopropyl)amine (H₂N(CH₂)₃NH(CH₂)₃NH₂), tris(2-aminoethyl)amine(N (CH₂CH₂NH₂)₃), 1,8-diaminooctane (H₂N(CH₂)₈NH₂), 1,10-diaminodecane(H₂N(CH₂)₁₀NH₂), and 1,12-diaminododecane (H₂N(CH₂)₁₂NH₂), can also beused.

[0052] N6-(Ï

-amino-linker) adenosine 5′-diphosphate and N6-(Ï

-amino-linker) adenosine 5′-triphosphate can be prepared by contacting6-chloropurine riboside 5′-diphosphate or 6-chloropurine riboside5′-triphosphate with a diamine to produce an N6-(Ï

-amino-linker) adenosine 5′-diphosphate or N6-(llamino-linker) adenosine5′-triphosphate. Diamines may include ethylenediamine (EDA),1,4-butanediamine (BDA), 1,6-hexanediamine (HDA), 2,2′-oxydiethylamine(ODEA), 2,2′-(ethylenedioxy)diethylamine (EDODEA), and4,7,10-trioxa-1,13-tridecanediamine (TOTDDA). These diamines providelinkers of (CH₂)₂, (CH₂)₄, (CH₂)₆, C₂H₄OC₂H₄, and C₃H₆OC₂H₄OC₂H₄OC₃H₆,respectively. Additional diamines such as spermidine (NH₂(CH₂)₃NH(CH₂)₄NH₂), spermine, (NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NH₂), polyethylene glycoldiamines (H₂N-PEG—NH₂), 1,4-bis(3-aminopropoxy)butane(H₂N(CH₂)₃O(CH₂)₄O(CH₂)₃NH₂), bis(3-aminopropyl)amine(H₂N(CH₂)₃NH(CH₂)₃NH₂), tris(2-aminoethyl) amine (N(CH₂CH₂NH₂)₃),1,8-diaminooctane (H₂N(CH₂)₈NH₂), 1,10-diaminodecane (H₂N(CH₂)₁₀NH₂),and 1,12-diaminododecane (H₂N(CH₂)₁₂NH₂), can also be used.

[0053] N6-(Ï

-sulfhydryl-linker) adenosine 5′-monophosphate molecules,

[0054] N6-(Ï

-sulfhydryl-linker) adenosine 5′-diphosphate molecules, and

[0055] N6-(Ï

-sulfhydryl-linker) adenosine 5′-triphosphate molecules can be preparedby contacting an aminothiol with 6-chloropurine riboside5′-monophosphate, 6-chloropurine riboside 5′-diphosphate or6-chloropurine riboside 5′-triphosphate to produce an N6-(Ï

- sulfhydryl-linker) adenosine 5′-monophosphate, N6-(Ï

-sulfhydryl-linker) adenosine 5′-diphosphate, or N6-(Ï

-sulfhydryl-linker) adenosine 5′-triphosphate.

[0056] N6-(Ï-amino-linker) adenosine 5′-O-thiomonophosphate, N6-(Ï

-amino-linker) adenosine 5′-[Î²-thio]diphosphate, and N6-(Ï

-amino-linker) adenosine 5′-[Î³-thio]triphosphate can be prepared bycontacting 6-chloropurine riboside 5′-O-thiomonophosphate,6-chloropurine riboside 5′-[Î²-thio]diphosphate, or 6-chloropurineriboside 5-[Î³-thio]triphosphate with a diamine to produce an N6-(Ï

-amino-linker) adenosine 5′-O-thiomonophosphate, N6-(Ï

-amino-linker) adenosine 5′-[Î²-thio]diphosphate, or N6-(Ï

-amino-linker) adenosine 5′-[Î²-thio]diphosphate. Diamines may includeethylenediamine (EDA), 1,4-butanediamine (BDA), 1,6-hexanediamine (HDA),2,2′-oxydiethylamine (ODEA), 2,2′-(ethylenedioxy)diethylamine (EDODEA),and 4,7,10-trioxa-1,13-tridecanediamine (TOTDDA). These diamines providelinkers of (CH₂)₂, (CH₂)₄, (CH₂)₆, C₂H₄OC₂H₄, and C₃H₆OC₂H₄OC₂H₄OC₃H₆,respectively. Additional diamines such as spermidine(NH₂(CH₂)₃NH(CH₂)₄NH₂), spermine, (NH₂ (CH₂)₃NH(CH₂)₄NH(CH₂)₃NH₂),polyethylene glycol diamines (H₂N-PEG—NH₂),1,4-bis(3-aminopropoxy)butane (H₂N(CH₂)₃O (CH₂)₄O(CH₂)₃NH₂),bis(3-aminopropyl)amine (H₂N(CH₂)₃NH (CH₂)₃NH₂), tris(2-aminoethyl)amine(N(CH₂CH ₂NH₂)₃), 1,8-diaminooctane (H₂N(CH₂)₈NH₂), 1,10-diaminodecane(H₂N(CH₂) ₁₀NH₂), and 1,12-diaminododecane (H₂N(CH₂)₁₂NH₂), can also beused.

[0057] These N6-(Ï

-amino-linker) adenosine 5′-monophosphate molecules can be genericallyrepresented by structure (VI) in FIG. 2. In the structure, linker L cangenerically be any alkyl, alkoxy, alkoxy alkyl, alkylamine, orpolyalkylamine group (such as described above). Specific examplesinclude N6-aminoethyl adenosine 5′-monophosphate (N6-EDA-AMP),N6-(4-aminobutyl) adenosine 5′-monophosphate (N6-BDA-AMP),N6-(6-aminohexyl) adenosine 5′-monophosphate (N6-HDA-AMP),N6-(2-aminoethyl) adenosine 5′-monophosphate (N6-ODEA-AMP),N6-(3,6-dioxa-8-aminooctyl) adenosine 5′-monophosphate (N6-EDODEA-AMP),and N6-(4, 7, 10-trioxa-13-aminotridecyl) adenosine 5′-monophosphate(N6-TOTDDA-AMP).

[0058] The N6-(Ï

-amino-linker) adenosine 5′-monophosphate molecules can be furtherreacted with an activated group to modify the linker terminal aminogroup. For example, succinimide esters can be used to add additionalfunctional moieties. One example of this concept is reaction with5(6)-carboxyfluorescein N-hydroxysuccinimide ester (5(6)-FAM-SE) toproduce fluorescein derivatives. Carboxyfluorescein is commonly obtainedas a mixture of 5- and 6- isomers, although either can be usedindividually. A list of example products includes N6-FAM-EDA-AMP,N6-FAM-BDA-AMP, N6-FAM-HDA-AMP, N6-FAM-ODEA-AMP, N6-FAM-EDODEA-AMP, andN6-FAM-TOTDDA-AMP. Similarly, biotin N-hydroxysuccinimide ester(biotin-SE) can be used to produce biotinylated derivatives. A list ofexample products include N6-biotin-BDA-AMP, N6-biotin-HDA-AMP,N6-biotin-EDODEA-AMP, and N6-biotin-TOTDDA-AMP.

[0059] The N6-(Ï

-amino-linker) adenosine 5′-diphosphate molecules and N6-(Ï

-amino-linker) adenosine 5′-triphosphate molecules can be furtherreacted with an activated group to modify the linker terminal aminogroup. For example, N-hydroxysuccinimide esters can be used to addadditional functional moieties. One example of this concept is reactionwith 5(6)-carboxyfluorescein N-hydroxysuccinimide ester (5(6)-FAM-SE) toproduce fluorescein derivatives.

[0060] The N6-(Ï

-sulfhydryl-linker) adenosine 5′-monophosphate molecules, N6-(Ï

-sulfhydryl-linker) adenosine 5′-diphosphate molecules, and N6-(Ï

-sulfhydryl-linker) adenosine 5′-triphosphate molecules can be furtherreacted with an activated group to modify the linker terminal thiolgroup. For example, haloacetamide, halide, and maleimide derivatives canbe used to add additional functional moieties. One example of thisconcept is reaction with fluorescein maleimide, or fluoresceinbromoacetamide to produce fluorescein derivatives of adenosine. Biotinderivatives of maleimide or haloacetamide can be used to producebiotinylated derivatives of adenosine.

[0061] The N6-(Ï

-amino-linker) adenosine 5′-O-thiomonophosphate, N6-(Ï

-amino-linker) adenosine 5′-[Î²-thio]diphosphate, and N6-(Ï

-amino-linker) adenosine 5′-[Î³-thio]triphosphate can also be reactedwith an activated group to modify the thiophosphate group. For example,haloacetamide, halide, and maleimide derivatives can be used to addadditional functional moieties. One example of this concept is reactionwith fluorescein maleimide, or fluorescein bromoacetamide to producefluorescein derivatives of adenosine. Biotin derivatives of maleimide orhaloacetamide can be used to produce biotinylated derivatives ofadenosine.

[0062] AMP 5′-derivatives of amino acids, neurotransmitters (such asserotonin, dopamine, epinephrine (adrenalin), tyamine, histamine), aminoacid-sugars (such as glucose cysteine) peptides, and proteins can beprepared by contacting AMP with the amino-containing compound (carboxygroups preferably should be protected to avoid reaction with EDAC) andEDAC to produce a 5′-amino acid-, neurotransmitter-, amino acid-sugar-,peptide-, or protein-derivative of AMP.

[0063] AMP, ADP, and ATP N6-derivatives of amino acids,neurotransmitters (such as serotonin, dopamine, epinephrine (adrenalin),tyamine, histamine), amino acid-sugars (such as glucose cysteine)peptides, and proteins can be prepared by contacting AMP, ADP, and ATPwith the amino-containing compound to produce an N6- amino acid-,neurotransmitter-, amino acid-sugar-, peptide-, or protein- derivativeof AMP, ADP, or ATP.

[0064] Kits

[0065] An additional embodiment of the invention is related to kitsuseful for the preparation of the above described RNA molecules. Thekits can comprise reagents, enzymes, nucleic acid templates, one or morecontainers, buffers, solvents, instruction protocols, purificationmaterials, positive and negative controls, standards, and so on.

[0066] An example of such a kit can comprise a RNA polymerase enzyme,one or more of the previously discussed adenosine derivatives, ATP, UTP,GTP, and CTP.

[0067] Methods of Preparation of RNA Molecules

[0068] Another aspect of the invention relates to methods of preparingRNA molecules having adenosine derivatives or coenzymes at their 5′terminal end. Generally, the method comprises providing a DNA templatecomprising a RNA polymerase promoter sequence; contacting the DNAtemplate, an RNA polymerase enzyme, an adenosine derivative, ATP, UTP,GTP, and CTP to prepare a reaction mixture; and incubating the reactionmixture under conditions suitable for RNA transcription to prepare RNAmolecules. It is presently preferred that the method is an in vitroenzymatic method.

[0069] The DNA template can generally be any DNA template. The DNAtemplate can be single stranded or double stranded. The length of theDNA sequence can generally be any number of bases or base pairs. Forexample, the DNA sequence can be at least about 5 bases, at least about10 bases, at least about 20 bases, at least about 30 bases, at leastabout 40 bases, at least about 50 bases, at least about 60 bases, atleast about 70 bases, at least about 80 bases, at least about 90 bases,at least about 100 bases, at least about 200 bases, at least about 300bases, at least about 400 bases, at least about 500 bases, at leastabout 600 bases, at least about 700 bases, at least about 800 bases, atleast about 900 bases, at least about 1000 bases, at least about 2000bases, and so on. There is no practical upper limit to the length of theDNA sequence. The DNA sequence can be a single DNA sequence, or amixture of DNA sequences.

[0070] It is presently preferred that the promoter sequence be the T7Class II promoter sequence (5′-TMTACGACTCACTATTAGGAG-3′; SEQ ID NO:7).It is currently preferred that the RNA polymerase enzyme is the T7 RNApolymerase enzyme.

[0071] The adenosine derivative can be any adenosine derivative orcoenzyme discussed above. The reaction mixtures typically will containATP, UTP, GTP, and CTP in order to produce a transcribed RNA moleculecontaining A, U, G, and C. It is possible that some DNA templates maylack one or more of the four bases. In such a case, one or more of ATP,UTP, GTP, and CTP could be omitted. For example, if the DNA template tobe transcribed contained only A, G, and C (i.e. no T), then UTP could beomitted from the reaction mixture as the transcribed RNA would containonly A, G, and C.

[0072] The reaction mixture can further comprise buffers, surfactants,salts, RNase inhibitors, and other common molecular biology reagents.Incubation under conditions suitable for RNA transcription largelyinvolves selection of buffer, pH, time, and temperature conditionsaccording to the activity requirements of the RNA polymerase enzymeaccording to the manufacturer's protocols. For example, T7 RNApolymerase can be used with a solution of 40 mM Tris (pH 8.0), 6 mMMgCl₂, 2 mM spermidine, 0.01% Triton X-100, 5 mM DTT, and 0.2 unit/Î¼LRNase inhibitor. Incubation can be performed for about two hours toabout four hours at a temperature of about 37 Â° C., or according to theenzyme manufacturer's protocols.

[0073] In order to prepare a radiolabelled RNA molecule, the reactionmixture can further comprise radioactive components such as [Î±-³²P]ATP,[Î±-³²P]GTP, [Î±-³²P]CTP, [Î±-³²P]UTP, [Î±-³⁵S]ATP, [Î±-^(S)]GTP,[Î±-³⁵S]CTP, and [Î±-³⁵S]UTP.

[0074] The method can further comprise covalently attaching amino acids,neurotransmitters (such as serotonin, dopamine, epinephrine (adrenalin),tyamine, histamine), amino acid-sugars (such as glucose cysteine)peptides, and proteins to the RNA molecule as discussed above. Theattaching step can be performed on the adenosine derivative prior to itsincorporation into the transcribed RNA molecule, or after itsincorporation. It is presently preferred that the attaching step beperformed on the adenosine derivative prior to its incorporation intothe transcribed RNA molecule.

[0075] The method can further comprise one or more purification stepsafter the incubation step. The purification step can involve use of gelelectrophoresis (such as polyacrylamide gel electrophoresis; PAGE),membrane filtration, or liquid chromatography. The method can furthercomprise visualizing and/or quantifying the prepared RNA molecules usingfluorescence, phosphorimaging, or other radioimaging methods.

[0076] Methods of Use

[0077] The above described RNA molecules can be used in a wide array ofchemical and biological applications. The RNA molecules can be used fornucleic acid detection, hybridizing to a complementary DNA sequence. TheRNA molecules can be used in the designed or random generation ofcatalytic RNAs. The RNA molecules can be used in antisense applications.The RNA molecules can be used to study the structure and function of RNAsequences. The RNA molecules can be used to investigate interactionbetween RNA and other biomolecules such as protein and amino acids,polysaccharides and sugars, lipods, coenzymes, nucleotides, and RNA.

[0078] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the scope of theinvention.

EXAMPLES Example 1 Materials

[0079] 3′-Dephospho coenzyme A (De-P-CoA), NAD, and FAD were purchasedfrom Sigma-Aldrich (St. Louis, Mo.) (Catalog # D3385, N6522, and F6625for De-P-CoA, NAD, and FAD, respectively). ATP, UTP, GTP, and CTP werefrom Boehringer Mannheim (Indianapolis, Ind.). T7 RNA polymerase camefrom Epicentre Technologies (Madison, Wis.). RNase inhibitor, Taq DNApolymerase, nuclease P1, DTT, dATP, dTTP, dGTP, and dCTP were obtainedfrom Promega Life Science Technologies (Madison, Wis.). ThiopropylSepharose 6B was from Amersham Pharmacia Biotech (Piscataway, N.J.). DNAoligonucleotides were obtained from either Operon Technologies (Alameda,Calif.) or Integrated DNA Technologies (Coralville, Iowa).

[0080] The following chemicals were from Sigma-Aldrich-Fluka (St. Louis,Mo.): Ethylenediamine (EDA), Sigma E4379, 1,6-Hexanediamine (HDA), SigmaH2381, Biotin N-hydroxysuccinimide ester (Biotin-SE), Sigma H1759,5(6)-Carboxyfluorescein N-hydroxysuccinimide ester [5(6)-FAM-SE], SigmaC1609, N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride(EDAC), Sigma E7750, 6-Chloropurine riboside, Sigma C8276,8-(6-Aminohexyl) aminoadenosine 5′-monophosphate lithium salt(8-HDA-AMP), Sigma A3771. 1,4-Butanediamine (BDA), Fluka 32791,2,2′-Oxydiethylamine dihydrochloride (ODEA), Fluka 75961,2,2′-(Ethylenedioxy)diethylamine (EDODEA), Fluka 03739,4,7,10-Trioxa-1,13-tridecanediamine (TOTDDA), Fluka 92892, Phosphorusoxychloride, Fluka 79580, Triethyl phosphate, Aldrich T6110-7.

Example 2 Transcription, DNazyme Cleavage, and Gel Electrophoresis

[0081] Transcription promoter sequence was derived from the T7 class IIpromot J. J., and Studier, F. W., J. Mol Biol. 166: 477-535 (1983)). TheRNA seq previously isolated coenzyme-synthesizing ribozyme (Huang, F.,et al., Bi 15555 (2000)), CoES7, with the sequence of5′-AGGGMGTGCTACCACMCUUUAGCCAUMUGUCACUUCUGCCGCG 3′ (SEQ ID NO:1).Transcription was carried out under the following stand of ATP, UTP,GTP, and CTP, 40 mM Tris (pH 8.0), 6 mM MgCl₂, 2 mM sp 100, 5 mM DTT,0.2 unit/mL RNase inhibitor, 0.2 mM DNA templates, and polymerase. Inaddition, different concentrations of coenzymes, De-P-CoA included toinitiate transcription. For quantitation purposes, [Î±-³²P]ATP (N MA)was also added to transcription solutions to internally label RNA transreactions were allowed to proceed for 2 hours at 37 Â° C. before producta PAGE.

[0082] To physically separate CoA-RNA from pppRNA by PAGE, a differentRNA 35 nucleotides was designed to allow a specific DNazyme (Santoro, S.W. G. F., Proc. Natl. Acad. Sci. USA 94: 4262-4266 (1997)) cleavageafter tra Two DNA oligonucleotides with the sequence of5′-CAGTTMTACGACTCACTATTAGGGMGCGGGCATGCGGCCAGCCAT 3′ (SEQ ID NO:2) and5′-TGATCGGCTATGGCTGGCCGCATGCCCG-3′ (were used to construct (by PCR) aDNA duplex template, from which a 35 sequence of5′-AGGGMGCGGGCAUGCGGCCAGCCAUAGCCGAUCA-NO:4) was transcribed. A DNazyme(with the sequence of 5′-TGATCGGCTAGGCTAGCTACMCGAGGCTGGCCGC-3′; SEQ IDNO:5 used to specifically cleave off a portion of the 3′ heterogeneousends of bo and pppRNA, yielding a homogeneous length of 25 nt CoA-RNAand pppR transcription conditions were used with the exception ofdifferent concentr Transcribed RNA (about 2 mM) was incubated with 0.4mM DNazyme for 37 Â° C. in 50 mM MgCl₂ and 50 mM Tris, pH 8.0. RNA wasthen analyzed denaturing PAGE.

Example 3 RNA Quantitation

[0083] After polyacrylamide gel electrophoresis, individual RNA bandswere visualized through phosphorimaging (Molecular Imager, Bio-RadLaboratories, Hercules, Calif.). Quantitation of different RNA bands wasachieved through profile integration after horizontal backgroundsubtraction. Coenzyme-RNA (CoE-RNA) yields were calculated based onrelative intensity of pppRNA bands (N, N+1, and N+2 bands) in theabsence of coenzymes. To confirm the validity of the calculation,DNazyme-cleaved pppRNA and CoA-RNA were quantitated directly after gelresolution of CoA-RNA from pppRNA. In addition, CoA-RNA yield wasdetermined independently by thiopropyl Sepharose 6B affinitychromatography based on its free sulfhydryl group.

Example 4 Fluorescence Emission Spectra of FAD-RNA

[0084] FAD-RNA was prepared by transcription under the standardconditions in the presence of 4 mM FAD (no [Î±-³²P]ATP was added). Twoconsecutive purification procedures were used to ensure that purifiedFAD-RNA does not contain free FAD from the transcription solution. After4 hours of transcription at 37 Â° C., the RNA sample was loaded onto aMicrocon filter (M30, Millipore Corp., Bedford, Mass.) and centrifugedat 14,000 g to a final volume of about 5 mL. Next, 200 mL of water wasadded to the Microcon filter and centrifuged down to about 5 mL solutionleft on the filter. Equal volume of gel loading solution containing 7 Murea and 0.02% xylene cyanol was added to the filter. Recovered RNA fromthe filter was heated at 90 Â° C. for 1 minute and loaded onto an 8%denaturing polyacrylamide gel. After gel electrophoresis, RNA bands(containing both pppRNA and FAD-RNA) were located by UV shadowing andexcised from the gel. RNA was then eluted and recovered by ethanolprecipitation.

[0085] Fluorescence emission spectra of 0.1 mM free FAD and 0.1 mMFAD-RNA (after FAD-RNA yield correction) were recorded with an ISS PCdfluorometer (ISS Inc., Champaign, Ill.). FAD has two absorption peaks,located at approximately 370 nm and 450 nm (with e450>e370),respectively. Its fluorescence emission spectrum peaks at about 530 nm.When FAD is excited at 450 nm, the Raman scatter peak co-localizes withfluorescence emission peak at about 530 nm. To avoid the interference ofthe Raman scatter with fluorescence emission, both free FAD and FAD-RNAwere excited at 370 nm. To see the effect of RNA on FAD fluorescenceemission intensity, FAD-RNA emission spectra were also recorded beforeand after complete digestion by nuclease P1. Digestion of RNA wasachieved by adding 1 unit of nuclease P1 in a buffer containing 10 mMNaAc (pH 5.2) and 0.4 mM ZnCl₂. The effect of urea on FAD-RNAfluorescence was also investigated with two different concentrations ofurea, 4 M and 7 M.

Example 5 Adenosine Derivative-Initiated Transcription

[0086] ATP-initiated transcription (Huang, F., et al., Biochemistry, 39:15548-15555 (2000)) under the T7 class II promoter (Dunn, J. J., andStudier, F. W., J. Mol Biol. 166: 477-535 (1983)) has been previouslydemonstrated. As an extension of the work, we sought to develop ageneral transcription method for preparation of RNA with adenosinederivatives linked to its 5′ end. Because many biological cofactors,such as coenzymes CoA, NAD, and FAD, contain an adenosine group, theymay be used as the transcription initiators under the T7 class IIpromoter, leading to the synthesis of cofactor-linked RNA. Because CoAcontains a 3′ phosphate group that can block 3′ extension,dephosphorylated CoA (De-P-CoA) must be used to initiate transcription.

[0087] Transcription of CoES7 RNA was carried out under the standardconditions, with the addition of various concentrations of coenzymes.After fractionation by PAGE, different RNA bands became clearly visible.In the absence of any coenzymes (lane 1), transcription produced threepppRNA bands in decreasing order: the full-length RNA transcripts (Nband), N+1, and N+2 bands (with one and two extra nucleotides added atthe 3′ end of full-length RNA). Their relative yields were 59%, 35%, and6% (from N to N+2), respectively. When one of the three coenzymes,De-P-CoA, NAD, and FAD, was added to the transcription solution (lanes2-4), no additional RNA bands were generated. However, the relativeintensity of the three RNA bands in each lane changed considerably whencompared with that of lane 1. In all three lanes (lanes 2-4), theintensity of the N band RNA decreased, while both N+1 and N+2 bands weresignificantly higher than those in lane 1. In the presence of 4 mMDe-P-CoA (lane 2), the relative intensity of the three bands changed to40%, 45%, and 15% (from N to N+2). The relative RNA band intensity were30%, 49%, and 21% for both NAD- and FAD-initiated transcription (lanes 3and 4). These changes in RNA band relative intensity resulted from theincorporation of coenzymes (De-P-CoA, NAD, and FAD) into RNA. When oneof these coenzymes is incorporated to the 5′ end of RNA, thecoenzyme-linked RNA (CoE-RNA) behaves as if an extra normal nucleotideis added to the pppRNA. Therefore, the N band CoE-RNA co-migrates withthe N+1 band pppRNA, while the N+1 band CoE-RNA and the N+2 band pppRNArun together in the gel. The N+2 band CoE-RNA was barely visible becauseof its low intensity. To confirm the incorporation of coenzymes into RNAunder the class 11 promoter, three independent experiments wereperformed: resolution of CoA-RNA from pppRNA by gel after DNazymecleavage, measurement of free sulfhydryl group of CoA-RNA, andfluorescence emission spectrum of FAD-RNA.

[0088] Both the total RNA yields and CoE-RNA yields depend on coenzymeconcentrations, as shown in FIG. 4A-4C. Among the three coenzymesincluded in this investigation, individual coenzymes appear to have somedifferent effects on transcription. For De-P-CoA incorporation (FIG.4A), the total RNA yield (pppRNA+CoA-RNA) first increases slightly withthe concentration of De-P-CoA, indicating that De-P-CoA stimulatestranscription to some extent at low concentrations. However, at higherconcentrations, the stimulatory effect of De-P-CoA disappears, andeventually becomes inhibitory. On the other hand, the CoA-RNA yieldincreases when higher concentrations of De-P-CoA are added to thetranscription solution. CoA-RNA reaches 30-35% of total RNA synthesizedat 4-8 mM De-P-CoA. Both NAD and FAD have a similar effect on relativeCoE-RNA yields (FIGS. 4B and 4C). NAD-RNA and FAD-RNA first increaserapidly with the concentration of NAD and FAD, and then plateau at highNAD and FAD concentrations. In the presence of 4-8 mM NAD or FAD,transcription produces 35-40% NAD-RNA or FAD-RNA over the total RNAsynthesized. On the other hand, NAD and FAD appear to exert differenteffects on total RNA yields. While different concentrations of NADslightly stimulate transcription (FIG. 4B), FAD appears to have somewhatinhibitory effect, especially at high concentrations (>4 mM) (FIG. 4C).Results in FIGS. 4A-4C were derived from two independent transcriptionexperiments. The relative CoE-RNA yields in FIGS. 4A-4C are veryreproducible. However, some degree of variation in total RNA yields mayexist among different experiments. In the presence of higher than 8 mMcoenzymes (especially De-P-CoA and FAD), total RNA yields can besignificantly lower than under the standard transcription conditions.

Example 6 Resolution of Coenzyme-RNA from pppRNA

[0089] To physically resolve coenzyme-RNA from pppRNA by gel, a DNazyme(Santoro, S. W., and Joyce, G. F., Proc. Natl. Acad. Sci. USA, 94:4262-4266 (1997)) was designed to trim off the 3′ heterogeneous ends ofRNA. Although transcription produces N, N+1, and N+2 bands of bothpppRNA and CoA-RNA, the DNazyme cuts at a specific site and yields bothCoA-RNA and pppRNA of the same length of 25 nt. Comparing lanes 1 and 2,the upper band in lane 2 represents CoA-RNA (33%), while the bottom bandis pppRNA (it is not clear why there was a weak band above the expectedproduct bands, but it represented well below 10% of total RNA). TheCoA-RNA yield agrees well (within the experimental error of 5%) with theresults from FIG. 4A. When the concentration of De-P-CoA was maintainedat 4 mM and the ATP concentration was reduced from 1 mM to 0.5 mM, 0.2mM, and 0.1 mM (keeping GTP, CTP, and UTP concentrations at 1 mM), therelative CoA-RNA yield increased from 33% to 46%, 52%, and 56%. On theother hand, the total RNA yield decreased from 110% (relative to the RNAobtained at 1 mM ATP and 0 mM De-P-CoA) to 85%, 44%, and 12%. Theseemingly more intense bands in lanes 3 & 4 relative to lane 2 resultedfrom higher ratios of [Î±-³²P]ATP/ATP when ATP concentration waslowered.

[0090] The effect of 3′ blocked adenosine derivatives on transcriptionwas also investigated by gel after DNazyme cleavage of RNA. At 4 mM,neither CoA nor 3′ AMP has detectable effect on either normal RNAtranscription (with no coenzymes) or CoA-RNA transcription (in thepresence of 4 mM De-P-CoA). The result is in excellent accordance withthe specific adenosine derivative-initiated transcription, whichrequires a free 3′ hydroxyl group for RNA extension.

[0091] To further confirm the yields of CoE-RNA derived from PAGE andphosphorimaging analysis, the CoA-RNA yield was independently determinedthrough its 5′ free sulfhydryl group by thiopropyl Sepharose 6B affinitychromatography. CoES7 RNA was prepared under 1 mM ATP and 4 mM De-P-CoAand purified by membrane filtration with Microcon 30. After passing theRNA through a thiopropyl Sepharose 6B column followed by thoroughwashing with water, 30-35% of loaded RNA was retained. The retained RNAwas completely released from the column by 30 minutes incubation with 20mM DTT in 20 mM Tris, pH 8.5. The result not only confirms that COA-RNAis synthesized by T7 RNA polymerase in the presence of De-P-CoA but alsocorroborates quantitatively the results from PAGE and phosphorimaginganalysis. Further confirmation of CoE-RNA synthesis comes from thefollowing fluorescence measurement of FAD-RNA.

Example 7 Fluorescence Emission Spectra

[0092] Fluorescence emission spectra of purified FAD-RNA (CoES7) andfree FAD were determined. Except for lower fluorescence intensity ofFAD-RNA relative to that of free FAD, both spectra are similar, with noapparent spectral shift. At the same concentration, fluorescenceintensity of FAD-RNA is about 35-40% of that of free FAD in 20 mM MES,pH 6.0. This decrease in fluorescence of FAD upon covalent coupling toRNA is presumably due to quenching by FAD-linked RNA, because similarconcentrations of RNA added to free FAD solutions (0.1 mM) do not affectthe fluorescence emission of FAD.

[0093] To demonstrate the effect of covalent linkage to RNA, emissionspectra of FAD-RNA were taken before and after complete digestion ofFAD-RNA by nuclease P1. Although no spectral shift was observed,fluorescence intensity increased 2.5 fold to the level of free FAD. Thisresult confirms that covalent linkage to RNA considerably reduces thefluorescence quantum yield of FAD.

[0094] The effect of covalent coupling to RNA can be furtherdemonstrated through FAD-RNA denaturation by urea. High concentrationsof urea significantly increase the fluorescence intensity of FAD-RNA.

[0095] In 4 M and 7 M urea (containing 0.5 M sodium acetate, pH 5.2),fluorescence intensity of FAD-RNA was enhanced 2.5 and 5.1 fold,respectively. On the other hand, free FAD's fluorescence increased only1.7 and 2.7 fold under the same conditions.

[0096] These results suggest that the reduced fluorescence of FAD-RNArelative to that of free FAD is not a result of the covalent linkageitself between FAD and the 5′ end of RNA. The fluorescence decrease israther caused by folded RNA conformation. In free FAD, stacking betweenthe isoalloxazine and the adenine significantly quenches thefluorescence intensity of isoalloxazine (Bessey, O. A., et al., J. Biol.Chem. 1.80: 755-769 (1949); Weber, G., Biochem. J. 47:114-121 (1950)).In the presence of 7 M urea, unstacking of the isoalloxazine and theadenine of free FAD results in a fluorescence enhancement of 2.7 fold.Under the same conditions, FAD-RNA is denatured (both RNA conformationand the stacking of the isoalloxazine and the adenine of FAD moiety),leading to a 5.1 fold increase in fluorescence intensity. The largerincrease in fluorescence intensity of FAD-RNA over free FAD in thepresence of 7 M urea reflects the contribution of the folded RNAconformation, which is corroborated by the 2.5-fold fluorescencereduction from free FAD to FAD-RNA. Both FAD and FAD-RNA exhibit similarfluorescence quantum yields (same fluorescence intensity from'sameconcentrations of FAD and FAD-RNA) in 7 M urea, indicating that theisoalloxazine group in FAD and FAD-RNA is in similar chemicalenvironment under these conditions. Thus, introducing the covalent bondby itself between FAD and RNA does not appear to affect FADfluorescence. However, the covalent linkage is necessary to bridge theinteraction between FAD and RNA, because similar RNA concentrationsadded to free FAD solutions do not affect FAD fluorescence intensity.

Example 8 Synthesis of 5′ Amino-Derivatives of Adenosine Phosphoramidate

[0097] Direct coupling of diamines with AMP (Chu, B. C., et al., NucleicAcids Res. 11: 6513-6529 (1983)) by the water-soluble carbodiimide,EDAC, was used to synthesize a series of six 5′ amino-derivatives ofadenosine phosphoramidate, which differ by the linker length. Thereactions and structures are shown in FIG. 1. Briefly, 0.25 Î¼ mol ofAMP and 5.0 Î¼ mol of one of the 6 diamines (ethylenediamine,1,4-butanediamine, 1,6-hexanediamine, 2,2′-oxydiethylamine,2,2′-(ethylenedioxy) diethylamine, and4,7,10-trioxa-1,13-tridecanediamine) were dissolved in 1.0 mL water. Thesolution pH was adjusted to 6.0-6.5 with 6 N HCl. Solid EDAC(hydrochloride, 1.5 Î¼ mol) was then added to the AMP-diamine mixtureand the reaction was allowed to proceed for 2 hours at room temperaturewith constant stirring. Product yields were determined by reverse phaseHPLC analysis under the following eluting conditions (column, AlltechExpedite C18, 10×4.6 mm and follow rate, 1 mL/minute): 5% MeOH/95% 20 mMphosphate (pH 7.0) for AMP with EDA the reactions; 10% MeOH/90% 20 mMphosphate (pH 7.0) for the reactions of AMP with BDA, HDA, ODEA, andEDODEA; 15% MeOH/85% 20 mM phosphate for the AMP and TOTDDA reaction.

[0098] A semi-preparative HPLC column (Waters Delta Pak.C18, 300×7.8 mm)was used to purify all six 5′ amino-derivatives of adenosinephosphoramidate. For each product purification, the column was firstequilibrated in 100% water. All of the reaction mixture (1-2 mL) wasloaded onto the column, followed by washing with about 30 mL of water.The pure product was then eluted with 30% MeOH. Collected productsolutions were concentrated under vacuum to a final volume of about 0.5mL. Concentrations of purified 5′ amino-derivatives of adenosinephosphoramidate were determined by their absorbance at 260 nm, using amolar extinction coefficient of 15,000 M⁻¹ cm⁻¹.

Example 9 Synthesis of N6Amino-Derivatives of Adenosine 5′ Monophosphate

[0099] A series of six N6 amino-derivatives of AMP were synthesized bydiamine-displacement of 6-chloropurine riboside 5′ monophosphate, whichwas prepared by phosphorylation of 6-chloropurine riboside withphosphorus oxychloride: To 7.7 ml of triethylphosphate, 3 mmol of6-chloropurine riboside was suspended at 0 Â° C. Small fractions ofphosphorus oxychloride (0.56 mL, 6 mmol) were added over a period of 30minutes with constant stirring. The suspension was kept stirring at 0 Â°C. for additional 1.5 hours, when the white suspension became clearsolution. Water (2 mL) was added to hydrolyze the 5′ phosphoryl chlorideto 5′ monophosphate. The solution was then neutralized to pH 5 by NaOH.HPLC analysis (Alitech Expedite C18, 10×4.6 mm; 10% MeOH/90% 20 mMphosphate; and follow rate, 0.5 mL/minute) indicated near completeconversion of 6-chloropurine riboside to 6-chloropurine riboside 5′monophosphate. The reaction mixture was directly used for the synthesisof the following N6 amino-derivatives of AMP.

[0100] To 0.1 mmol of the above 6-chloropurine riboside 5′ monophosphatesolution, 1 mmol of diamines (EDA, BDA, HDA, ODEA, EDODEA, TOTDDA intheir free amine state) was added. After stirring at room temperaturefor 30 minutes, the solution was acidified to pH 3.5 by HCI. HPLCanalysis [Alltech Expedite C18, 10×4.6 mm; 100%-70% ammonium formate (pH3.2) and 0-30% MeOH; follow rate, 0.5 mL/minute] indicated 60-90%conversion of 6-chloropurine riboside 5′ monophosphate toN6-amino-derivatives of AMP.

[0101] Products were purified by semi-preparative HPLC under thefollowing conditions: Waters Delta Pak C18, 300×7.8 mm; follow rate, 2mL/minute. The reaction mixture was loaded onto the columnpre-equilibrated with 20 mM ammonium formate (pH 3.2). Products wereeluted with 30-50% MeOH. Collected product solutions were concentratedunder vacuum and re-injected back onto the semi-preparative column thatwas equilibrated in 100% water. After washing with 2030 mL of water, theproduct was eluted with 50% MeOH. After concentration of collectedproduct fractions, their concentrations were determined by absorbance at267 nm (Îμ=18,500 M⁻¹ cm⁻¹).

Example 10 Synthesis of 5′-and N6-Fluorescein Derivatives of AdenosinePhosphoramidate and AMP

[0102] Fluorescein-tagged nucleotides of 5′- and N6-amino-derivatives ofadenosine phosphoramidate and AMP were prepared by reacting these 5′-and N6-amino-derivatives with 5(6)-carboxyfluoresceinN-hydroxysuccinimide ester [5(6)-FAM SE] as following: mixing 5 Î¼L of0.4 M amino-derivatives of adenosine phosphoramidate and AMP and 5 Î¼Lof 1 M NaHCO₃ (pH 8.0) with 10 Î¼L of 0.2 M 5(6)-FAM SE solution(freshly made in DMSO). The reaction mixture was incubated for 30minutes at room temperature. HPLC analysis (Alltech Expedite C18, 10×4.6mm; 30%-40% MeOH & 70%-60% 20 mM KH₂PO₄, pH 4.6) showed 70 90%fluorescein conversion to a pair (5 and 6) of fluorescein-taggednucleotides.

[0103] Purification was performed by HPLC under the followingconditions: loading all the reaction solution in 25-40% MeOH and 75-60%KH₂PO₄. After 15-30 min, MeOH concentration was increased by 5%. Twofractions containing fluorescein nucleotides from the elution werecollected and concentrated under vacuum. Their concentrations weredetermined by absorbance at 492 nm in 20 mM phosphate buffer (pH 7.0),Îμ=82,000 M⁻cm⁻¹.

Example 11 Synthesis of 5′- and N6-Biotin Derivatives of AMP

[0104] Seven biotinyl compounds of 5′- and N6-amino-derivatives ofadenosine phosphoramidate and AMP were prepared by reaction of biotinN-hydroxysuccinimide ester with the amino derivatives. To a mixture of20 Î¼L of 0.4 M amino-derivative of adenosine phosphoramidate or AMP and10 Î¼L of 1 M NaHCO₃, 20 Î¼L of freshly made 0.2 M biotin—NHS solution(in DMSO) was added. The reaction mixture was allowed to incubate for 30minutes at room temperature. HPLC analysis (Alltech Expedite C18, 10×4.6mm; 30% MeOH/70% 20 mM KH₂PO₄, pH 4.6) showed 40-60% conversion ofamino-derivatives of adenosine phosphoramidate and AMP to biotin-taggednucleotides.

[0105] Purification was performed by HPLC under the followingconditions: loading all the reaction sample onto a Waters Delta Pak C18,150×3.9 mm, equilibrated in 100% water. After 5-10 minutes, the mobilephase was changed to 15-20% MeOH. One major fraction containing theproduct biotinyl nucleotide was eluted. After another 5-10 minutes, MeOHconcentration was increased to 35-40%. Another major fraction containingthe desired product was eluted. Collected biotin nucleotide solutionswere concentrated and their concentrations were determined by absorbanceat 260 nm, using Îμ=15,000 M⁻¹ cm⁻¹.

Example 12 Incorporation of 5′- and N6-Amino, Fluorescein, and BiotinDerivatives into RNA by in vitro transcription

[0106] For all RNA transcription experiments, the promoter sequence wasderived from the T7 class II promoter (Dunn, J. J. and Studier, F. W. J.Mol. Biol., 166: 477-535 (1983); Huang, F. Nucleic Acids Res. 31: e8(2003)). The RNA sequence was a 35 mer used in previously transcriptionstudies (Huang, F. Nucleic Acids Res. 31: e8 (2003)):5′-AGGGAAGCGGGCAUGCGGCCAGCCAUAGCCGAUCA-3′ (SEQ ID NO:4). Transcriptionwas carried out under the following conditions (final concentrations): 1mM each of UTP, GTP, and CTP,0.25 mM ATP, 40 mM Tris (pH 8.0), 6 mMMgCl₂, 2 mM spermidine, 0.01% Triton X-100, 5 mM DUT, 0.2 Î¼M DNAtemplates, and 5 unit/Î4L T7 RNA polymerase. In addition, differentconcentrations of a transcription initiator (5′-amino derivatives ofadenosine phosphoramidate, N6-amino derivatives of AMP, or theirfluorescein and biotin derivatives) was added. The radiolabel,[Î±-³²P]ATP (NEN Life Science, Boston, Mass.), was also included totranscription solutions to internally label RNA transcripts. Alltranscription reactions were incubated for 2 h at 37 Â° C. beforeproduct analysis by 8% denaturing PAGE.

Example 13 Posttranscriptional Fluorescein Labeling of 5′ Amino-RNA

[0107] Fluorescein labeling of 5′ NH₂-linker-RNA (prepared above asinternally ³²P-labeled RNA) was achieved by reaction with 5(6)carboxyfluorescein-NHS in a total volume of 2 Î¼L containing 0.5 MNaHCO₃ and 0.1 M carboxyfluorescein-NHS (freshly prepared in DMSO).Reactions were incubated for 30 minutes at room temperature. Agel-loading dye solution (8 Î¼L) was then added, and 1 Î¼L of sampleswas loaded onto an 8% denaturing gel for analysis.

Example 14 RNA Quantitation

[0108] After polyacrylamide gel electrophoresis, the intensity ofindividual RNA bands were quantitated through phosphorimaging (MolecularImager, Bio-Rad Laboratories, Hercules, Calif.). Different RNA bandswere analyzed through profile integration after horizontal backgroundsubtraction. Derivatized RNA (5′-amino, fluorescein, and biotin) yieldswere calculated based on relative intensity of pppRNA bands (N, N+1, andN+2 bands) in the absence of AMP derivatives. To confirm the results ofthe calculation, 5′-biotin-labeled RNA yields were determinedindependently by streptavidin gel shift assay.

Example 15 Coupling of Amino Acids, Peptides, and Proteins toTranscribed RNA Molecules

[0109] The free terminal primary amine group of the adenosine 5′-(Ï

-amino-linker) phosphoramidate and the N6-(Ï

-amino-linker) adenosine 5′-monophosphate (VI) can be used to covalentlyattach amino acids, peptides, and proteins to the transcribed RNAmolecules. Standard peptide synthesis chemistries can be used to form acovalent amide bond between the amino group and a carboxylate grouppresent in the amino acid, peptide, or protein. Example chemistriesuseful for forming amide bonds include HBTU/HOBT, HATU/HOAT, PyBOP/HOBT,and OPFP preactivated amino acids/HOBT.

[0110] All of the compositions and/or methods disclosed and claimedherein can be made and executed without undue experimentation in lightof the present disclosure. While the compositions and methods of thisinvention have been described in terms of preferred embodiments, it willbe apparent to those of skill in the art that variations may be appliedto the compositions and/or methods and in the steps or in the sequenceof steps of the methods described herein without departing from theconcept and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the scope and concept of the invention.

1 9 1 62 DNA Artificial Isolated from randomized sequence library. 1agggaagtgc taccacaacu uuagccauaa ugucacuucu gccgcgggca ugcggccagc 60 ca62 2 55 DNA Artificial Designed to allow DNazyme cleavage aftertranscription. 2 cagtaatacg actcactatt agggaagcgg gcatgcggcc agccatagccgatca 55 3 28 DNA Artificial Designed to allow DNazyme cleavage aftertranscription. 3 tgatcggcta tggctggccg catgcccg 28 4 35 RNA ArtificialRNA transcribed from SEQ ID NO2. 4 agggaagcgg gcaugcggcc agccauagccgauca 35 5 35 DNA Artificial Isolated from randomized sequence library.5 tgatcggcta ggctagctac aacgaggctg gccgc 35 6 23 DNA Bacteriophage T3 6aattaaccct cactaaaggg aga 23 7 22 DNA Bacteriophage T7 7 taatacgactcactattagg ag 22 8 23 DNA Bacteriophage SP6 misc_feature (22)..(22) n isa, c, g, or t 8 atttaggtga cactatagaa gng 23 9 22 DNA Bacteriophage T7 9ctcctaatac tgagtcgtat ta 22

1. An RNA molecule containing an adenosine derivative covlently attachedat the 5′ end of the RNA molecule.
 2. The RNA molecule of claim 1,wherein the adenosine derivative is a 5′ derivative of adenosine or anN6 derivative of adenosine.
 3. The RNA molecule of claim 1, wherein theadenosine derivative is coenzyme A, FAD, NAD, S-(5′-adenosyl)-cysteine,S-(5′-adenosyl)-homocysteine, or S-(5-adenosyl)-methionine.
 4. The RNAmolecule of claim 1, wherein the adenosine derivative is a fluoresceinderivative of adenosine or a biotin derivative of adenosine.
 5. The RNAmolecule of claim 1, wherein the adenosine derivative is an adenosine5′-amino-linker phosphoramidate.
 6. The RNA molecule of claim 5, whereinthe linker is an alkyl group, an alkoxy group, an alkoxy alkyl group, analklamine group, or a polyalkylamine group.
 7. The RNA molecule of claim1, wherein the adenosine derivative is adenosine 5′-aminoethylphosphoramidate (5′-EDA-AMP), adenosine 5′-(4-aminobutyl)phosphoramidate (5′-BDA-AMP), adenosine 5′-(6-aminohexyl)phosphoramidate (5′-HDA-AMP), adenbsine 5′-(2-aminoethoxyethyl)phosphoramidate (5′-ODEA-AMP), adenosine 5′-(3,6-dioxa-8-aminooctyl)phosphoramidate (5′-EDODEA-AMP), or adenosine5′-(4,7,10-trioxa-13-aminotridecyl) phosphoramidate (5′-TOTDDA-AMP). 8.The RNA molecule of claim 1, wherein the adenosine derivative isadenosine 5′-(5)carboxyfluorescein aminoethyl phosphoramidate(5′-(5)FAM-EDA-AMP), adenosine 5′-(6) carboxyfluorescein aminoethylphosphoramidate (5′-(6)FAM-EDA-AMP), adenosine 5′-(5)carboxyfluorescein(4-aminobutyl) phosphoramidate (5′-(5)FAM-BDA-AMP), adenosine 5′-(6)carboxyfluorescein (4-aminobutyl) phosphoramidate (5′-(6) FAM-BDA-AMP),adenosine 5′-(5)carboxyfluorescein (6-aminohexyl) phosphoramidate(5′-(5)FAM-HDA-AMP), adenosine 5′-(6)carboxyfluorescein (6-aminohexyl)phosphoramidate (5′-(6)FAM-HDA-AMP), adenosine 5′-(5) carboxyfluorescein(2-aminoethoxyethyl) phosphoramidate (5′-(5)FAM-ODEA-AMP), adenosine5′-(6)carboxyfluorescein (2-aminoethoxyethyl) phosphoramidate(5′-(6)FAM-ODEA-AMP), adenosine 5′-(5)carboxyfluorescein(3,6-dioxa-8-aminooctyl) phosphoramidate (5′-(5)FAM-EDOEA-AMP),adenosine 5′-(6)carboxyfluorescein (3,6-dioxa-8-aminooctyl)phosphoramidate (5′-(6)FAM-EDODEA-AMP), adenosine5′-(5)carboxyfluorescein (4,7,10-trioxa-13-aminotridecyl)phosphoramidate (5′-(5)FAM-TOTDDA-AMP), or adenosine5′-(6)carboxyfluorescein (4,7,10-trioxa-13-aminotridecyl)phosphoramidate (5′-(6)FAM-TOTDDA-AMP).
 9. The RNA molecule of claim 1,wherein the adenosine derivative is adenosine 5′-biotin (6-aminohexyl)phosphoramidate (5′-biotin-HDA-AMP), adenosine 5′-biotin(3,6-dioxa-8-aminooctyl) phosphoramidate (5′-biotin-EDODEA-AMP), oradenosine 5′-biotin (4,7,10-trioxa-13-aminotridecyl) phosphoramidate(5′-biotin-TOTDDA-AMP).
 10. The RNA molecule of claim 1, wherein theadenosine derivative is an N6-amino-linker adenosine 5′-monophosphate.11. The RNA molecule of claim 10, wherein the linker is an alkyl group,an alkoxy group, an alkoxy alkyl group, an alkylamine group, or apolyalkylamine group.
 12. The RNA molecule of claim 1, wherein theadenosine derivative is N6-aminoethyl adenosine 5′-monophosphate (N6-EDA-AMP), N6-(4-aminobutyl) adenosine 5′-monophosphate (N6-BDA-AMP),N6-(6-aminohexyl) adenosine 5′-monophosphate (N6-HDA-AMP),N6-(2-aminoethoxyethyl) adenosine 5′-monophosphate (N6-ODEA-AMP),N6-(3,6-dioxa-8-aminooctyl) adenosine 5′-monophosphate (N6-EDODEA-AMP),or N6-(4, 7, 10-trioxa-13-aminotridecyl) adenosine 5′-monophosphate(N6-TOTDDA-AMP).
 13. The RNA molecule of claim 1, wherein the adenosinederivative is N6-(5)carboxyfluorescein aminoethyl adenosine5′-monophosphate (N6-(5)FAM-EDA-AMP), N6-(6) carboxyfluoresceinaminoethyl adenosine 5′-monophosphate (N6-(6)FAM-EDA-AMP),N6-(5)carboxyfluorescein (4-aminobutyl) adenosine 5′-monophosphate(N6-(5)FAM-BDA-AMP), N6-(6)carboxyfluorescein (4-aminobutyl) adenosine5′-monophosphate (N6-(6)FAM-BDA-AMP), N6-(5) carboxyfluorescein(6-aminohexyl) adenosine 5′-monophosphate (N6-(5)FAM-HDA-AMP), N6-(6)carboxyfluorescein (6-aminohexyl) adenosine 5′-monophosphate(N6-(6)FAM-HDA-AMP), N6-(5) carboxyfluorescein (2-aminoethyl) adenosine5′-monophosphate (N6-(5)FAM-ODEA-AMP), N6-(6) carboxyfluorescein(2-aminoethyl) adenosine 5′-monophosphate (N6-(6)FAM-ODEA-AMP), N6-(5)carboxyfluorescein (3,6-dioxa-8-aminooctyl) adenosine 5′-monophosphate(N6-(5)FAM-EDODEA-AMP), N6-(6) carboxyfluorescein(3,6-dioxa-8-aminooctyl) adenosine 5′-monophosphate(N6-(6)FAM-EDODEA-AMP), N6-(5) carboxyfluorescein (4, 7,10-trioxa-13-aminotridecyl) adenosine 5′-monophosphate(N6-(5)FAM-TOTDDA-AMP), or N6-(6)carboxyfluorescein (4, 7,10-trioxa-13-aminotridecyl) adenosine 5′-monophosphate(N6-(6)FAM-TOTDDA-AMP).
 14. The RNA molecule of claim 1, wherein theadenosine derivative is an adenosine 5′-sulfhydryl-linkerphosphoramidate.
 15. The RNA molecule of claim 14, wherein the linker isan alkyl group, an alkoxy group, an alkoxy alkyl group, or an alkylaminegroup.
 16. The RNA molecule of claim 1, wherein the adenosine derivativeis adenosine 5′-mercaptoethyl phosphoramidate.
 17. The RNA molecule ofclaim 1, wherein the adenosine derivative is adenosine5′-(5)carboxyfluorescein thioethyl phosphoramidate or adenosine5′-(6)carboxyfluorescein thioethyl phosphoramidate.
 18. The RNA moleculeof claim 1, wherein the adenosine derivative is adenosine 5′-biotinthioethyl phosphoramidate.
 19. The RNA molecule of claim 1, wherein theadenosine derivative is an N6-sulfhydryl-linker adenosine5′-monophosphate.
 20. The RNA molecule of claim 19, wherein the linkeris an alkyl group, an alkoxy group, an alkoxy alkyl group, or analkylamine group.
 21. The RNA molecule of claim 1, wherein the adenosinederivative is N6-mercaptoethyl adenosine 5′-monophosphate.
 22. The RNAmolecule of claim 1, wherein the adenosine derivative isN6-(5)carboxyfluorescein thioethyl adenosine 5′-monophosphate orN6-(6)carboxyfluorescein thioethyl adenosine 5′-monophosphate.
 23. TheRNA molecule of claim 1, wherein the adenosine derivative is N6-biotinthioethyl adenosine 5′-monophosphate.
 24. The RNA molecule of claim 1,wherein the adenosine derivative is adenosine 5′-O-thiomonophosphate,adenosine 5′-[Î²-thio]diphosphate, or adenosine5′-[Î³-thio]triphosphate.
 25. The RNA molecule of claim 24, wherein theadenosine derivative is a fluorescein derivative of adenosine or abiotin derivative of adenosine.
 26. The RNA molecule of claim 1, whereinthe adenosine derivative is an adenosine5′-(Î±,Î²-methylene)diphosphate, adenosine 5′-(Î²,Î³-imido)triphosphate,adenosine 5′-triphosphate Î³-(1-(2-nitrophenyl)ethyl) ester, or5′-p-fluorosulfonylbenzoyl adenosine.
 27. The RNA molecule of claim 1,wherein the adenosine derivative is N6-biotin (4-aminobutyl) adenosine5′-monophosphate (N6-biotin-BDA-AMP), N6-biotin (6-aminohexyl) adenosine5′-monophosphate (N6-biotin-HDA-AMP), N6-biotin (3,6-dioxa-8-aminooctyl)adenosine 5′-monophosphate (N6-biotin-EDODEA-AMP), or N6-biotin (4, 7,10-trioxa-13-aminotridecyl) adenosine 5′-monophosphate(N6-biotin-TOTDDA-AMP).
 28. The RNA molecule of claim 1, furthercomprising an amino acid, a peptide, a neurotransmitter, an aminoacid-sugar, or a protein covalently attached to the adenosinederivative.
 29. A method for the preparation of a RNA moleculecontaining an adenosine derivative covalently attached at the 5′ end ofthe RNA molecule, the method comprising: providing a DNA templatecomprising a RNA polymerase promoter sequence; contacting the DNAtemplate, an RNA polymerase enzyme, an adenosine derivative, ATP, UTP,GTP, and CTP to prepare a reaction mixture; and incubating the reactionmixture under conditions suitable for RNA transcription to prepare theRNA molecule.
 30. The method of claim 29, wherein the promoter sequenceis SEQ ID NO:7.
 31. The method of claim 29, wherein the adenosinederivative is a 5′ derivative of adenosine or an N6 derivative ofadenosine.
 32. The method of claim 29, wherein the adenosine derivativeis coenzyme A, FAD, NAD, S-(5′-adenosyl)-cysteine,S-(5′-adenosyl)-homocysteine, or S-(5′-adenosyl)-methionine.
 33. Themethod of claim 29, wherein the adenosine derivative is adenosine5′-O-thiomonophosphate, adenosine 5′-[Î²-thio]diphosphate, adenosine5′-[Î³-thio]triphosphate, adenosine 5′-(Î±,Î²-methylene)diphosphate,adenosine 5′-(Î²,Î³-imido) triphosphate, adenosine 5′-triphosphateÎ³-(1-(2-nitrophenyl) ethyl) ester, or5′-p-fluorosulfonylbenzoyladenosine.
 34. The-method of claim 29,OLE_LINK1 wherein the adenosine derivative is a fluorescein derivativeof adenosine, a biotin derivative of adenosine, an Alexa derivative ofadenosine, a BODIPY derivative of adenosine, a Cy3 derivative ofadenosine, or a Cy5 derivative of adenosine.
 35. The method of claim 29,wherein the adenosine derivative is covalently attached to an aminoacid, a peptide, a neurotransmitter, an amino acid-sugar, or a protein.36. The method of claim 29, wherein the RNA polymerase enzyme is the T7RNA polymerase enzyme.
 37. The method of claim 29, further comprisingpurifying the RNA molecule after the incubating step.
 38. The method ofclaim 29, wherein the reaction mixture further comprises at least oneradiolabel selected from the group consisting of [Î±-³²P]ATP,[Î±-³²P]GTP, [Î±-³²P]CTP, [Î±-³²P]UTP, [Î±-³⁵S]ATP, [Î-³⁵S]GTP,[Î±-³⁵S]CTP, and [Î±-³⁵S]UTP.
 39. A kit comprising a RNA polymeraseenzyme, an adenosine derivative, ATP, UTP, GTP, and CTP.
 40. The kit ofclaim 39, wherein the adenosine derivative is a 5′ derivative ofadenosine or an N6 derivative of adenosine.
 41. The kit of claim 39,wherein the adenosine derivative is coenzyme A, FAD, NAD,S-(5′-adenosyl)-cysteine, S-(5′-adenosyl)-homocysteine, orS-(5′-adenosyl)-methionine.
 42. The kit of claim 39, wherein theadenosine derivative is adenosine 5′-O-thiomonophosphate, adenosine5′-[Î²-thio]diphosphate, adenosine 5′-[Î³-thio]triphosphate, adenosine5′-(Î±,Î²-methylene)diphosphate, adenosine 5′-(Î²,Î³-imido)triphosphate, adenosine 5′-triphosphate Î³-(1-(2-nitrophenyl) ethyl)ester, or 5′-p-fluorosulfonylbenzoyladenosine.
 43. The kit of claim 39,wherein the adenosine derivative is a fluorescein derivative ofadenosine, a biotin derivative of adenosine, an Alexa derivative ofadenosine, a BODIPY derivative of adenosine, a Cy3 derivative ofadenosine, or a Cy5 derivative of adenosine.
 44. The kit of claim 39,wherein the RNA polymerase enzyme is the T7 RNA polymerase enzyme. 45.The kit of claim 39, further comprising at least one radiolabel selectedfrom the group consisting of [Î±-³²P]ATP, [Î±-³²P]GTP, [Î±-³²P]CTP,[Î±-³²P]UTP, [Î±-³⁵S]ATP, [Î±-³²S]GTP, [Î±-³⁵S]CTP, and [Î±-³⁵S]UTP. 46.An adenosine derivative defined as an adenosine 5′-amino-linkerphosphoramidate or a 5′-sulfhydryl-linker phosphoramidate.
 47. Theadenosine derivative of claim 46, wherein the linker is an alkyl group,an alkoxy group, an alkoxy alkyl group, an alkylamine group, or apolyalkylamine group.
 48. The adenosine derivative of claim 46, whereinthe adenosine derivative is adenosine 5′-aminoethyl phosphoramidate(5′-EDA-AMP), adenosine 5′-(4-aminobutyl) phosphoramidate (5′-BDA-AMP),adenosine 5′-(6-aminohexyl) phosphoramidate (5′-HDA-AMP), adenosine5′-(2-aminoethoxyethyl) phosphoramidate (5′-ODEA-AMP), adenosine5′-(3,6-dioxa-8-aminooctyl) phosphoramidate (5′-EDODEA-AMP), adenosine5′-(4,7,10-trioxa-13-aminotridecyl) phosphoramidate (5′-TOTDDA-AMP), oradenosine 5′-mercaptoethyl phosphoramidate.
 49. The adenosine derivativeof claim 46, wherein the adenosine derivative is adenosine5′-(5)carboxyfluorescein aminoethyl phosphoramidate (5′-(5)FAM-EDA-AMP),adenosine 5′-(6)carboxyfluorescein aminoethyl phosphoramidate(5′-(6)FAM-EDA-AMP), adenosine 5′-(5) carboxyfluorescein (4-aminobutyl)phosphoramidate (5′-(5) FAM-BDA-AMP), adenosine 5′-(6)carboxyfluorescein(4-aminobutyl) phosphoramidate (5′-(6)FAM-BDA-AMP), adenosine5′-(5)carboxyfluorescein (6-aminohexyl) phosphoramidate(5′-(5)FAM-HDA-AMP), adenosine 5′-(6) carboxyfluorescein (6-aminohexyl)phosphoramidate (5′-(6) FAM-HDA-AMP), adenosine 5′-(5)carboxyfluorescein(2-aminoethoxyethyl) phosphoramidate (5′-(5)FAM-ODEA-AMP), adenosine5′-(6)carboxyfluorescein (2-aminoethoxyethyl) phosphoramidate(5′-(6)FAM-ODEA-AMP), adenosine 5′-(5) carboxyfluorescein(3,6-dioxa-8-aminooctyl) phosphoramidate (5′-(5)FAM-EDODEA-AMP),adenosine 5′-(6) carboxyfluorescein (3,6-dioxa-8-aminooctyl)phosphoramidate (5′-(6)FAM-EDODEA-AMP), adenosine 5′-(5)carboxyfluorescein (4,7,10-trioxa-13-aminotridecyl) phosphoramidate(5′-(5)FAM-TOTDDA-AMP), adenosine 5′-(6) carboxyfluorescein(4,7,10-trioxa-13-aminotridecyl) phosphoramidate (5′-(6)FAM-TOTDDA-AMP),adenosine 5′-(5) carboxyfluorescein thioethyl phosphoramidate, oradenosine 5′-(6)carboxyfluorescein thioethyl phosphoramidate.
 50. Theadenosine derivative of claim 46, wherein the adenosine derivative isadenosine 5′-biotin (6-aminohexyl) phosphoramidate (5′-biotin-HDA-AMP),adenosine 5′-biotin (3,6-dioxa-8-aminooctyl) phosphoramidate(5′-biotin-EDODEA-AMP), adenosine 5′-biotin(4,7,10-trioxa-13-aminotridecyl) phosphoramidate (5′-biotin-TOTDDA-AMP),or adenosine 5′ biotin thioethyl phosphoramidate.
 51. An adenosinederivative defined as an N6-amino-linker adenosine 5′-monophosphate or a5′-sulfhydryl-linker phosphoramidate.
 52. The adenosine derivative ofclaim 51, wherein the linker is an alkyl group, an alkoxy group, analkoxy alkyl group, an alkylamine group, or a polyalkylamine group. 53.The adenosine derivative of claim 51, wherein the adenosine derivativeis N6-aminoethyl adenosine 5′-monophosphate (N6-EDA-AMP),N6-(4-aminobutyl) adenosine 5′-monophosphate (N6-BDA-AMP),N6-(6-aminohexyl) adenosine 5′-monophosphate (N6-HDA-AMP),N6-(2-aminoethoxyethyl) adenosine 5′-monophosphate (N6-ODEA-AMP),N6-(3,6-dioxa-8-aminooctyl) adenosine 5′-monophosphate (N6-EDODEA-AMP),N6-(4, 7, 10-trioxa-13-aminotridecyl) adenosine 5′-monophosphate(N6-TOTDDA-AMP), or N6-mercaptoethyl adenosine 5′-monophosphate.
 54. Theadenosine derivative of claim 51, wherein the adenosine derivative isN6-(5)carboxyfluorescein aminoethyl adenosine 5′-monophosphate(N6-(5)FAM-EDA-AMP), N6-(6) carboxyfluorescein aminoethyl adenosine5′-monophosphate (N6-(6)FAM-EDA-AMP), N6-(5)carboxyfluorescein(4-aminobutyl) adenosine 5′-monophosphate (N6-(5)FAM-BDA-AMP),N6-(6)carboxyfluorescein (4-aminobutyl) adenosine 5′-monophosphate(N6-(6)FAM-BDA-AMP), N6-(5) carboxyfluorescein (6-aminohexyl) adenosine5′-monophosphate (N6-(5)FAM-HDA-AMP), N6-(6) carboxyfluorescein(6-aminohexyl) adenosine 5′-monophosphate (N6-(6)FAM-HDA-AMP), N6-(5)carboxyfluorescein (2-aminoethyl) adenosine 5′-monophosphate(N6-(5)FAM-ODEA-AMP), N6-(6) carboxyfluorescein (2-aminoethyl) adenosine5′-monophosphate (N6-(6)FAM-ODEA-AMP), N6-(5) carboxyfluorescein(3,6-dioxa-8-aminooctyl) adenosine 5′-monophosphate(N6-(5)FAM-EDODEA-AMP), N6-(6) carboxyfluorescein(3,6-dioxa-8-aminooctyl) adenosine 5′-monophosphate(N6-(6)FAM-EDODEA-AMP), N6-(5) carboxyfluorescein (4, 7,10-trioxa-13-aminotridecyl) adenosine 5′-monophosphate(N6-(5)FAM-TOTDDA-AMP), N6-(6)carboxyfluorescein (4, 7,10-trioxa-13-aminotridecyl) adenosine 5′-monophosphate(N6-(6)FAM-TOTDDA-AMP), N6-(5)carboxyfluorescein thioethyl adenosine5′-monophosphate, or N6-(6)carboxyfluorescein thioethyl adenosine5′-monophosphate.
 55. The adenosine derivative of claim 51, wherein theadenosine derivative is N6-biotin (4-aminobutyl) adenosine5′-monophosphate (N6-biotin-BDA-AMP), N6-biotin (6-aminohexyl) adenosine5′-monophosphate (N6-biotin-HDA-AMP), N6-biotin (3,6-dioxa-8-aminooctyl)adenosine 5′-monophosphate (N6-biotin-EDODEA-AMP), N6-biotin (4, 7,10-trioxa-13-aminotridecyl) adenosine 5′-monophosphate(N6-biotin-TOTDDA-AMP), or N6-biotin thioethyl adenosine5′-monophosphate.